The goal of this application is to address RFA-CA-12-015 PQA1: What is the molecular mechanism by which a drug that is chronically used for other indications protects against cancer incidence and mortality? Vitamin D (VD) is a widely and chronically used supplement for maintenance of bone health and for treatment of osteoporosis, but a growing body of evidence has indicated that VD has potent chemopreventive effects against colon cancer by unknown mechanisms. Thus the central question to be addressed is: What is the molecular mechanism by which Vitamin D prevents colon cancer? Colon cancer is a leading cause of cancer- related deaths in the US. Low VD status is associated with increased colon cancer incidence, chemopreventive effects of VD have been demonstrated in colon cancer models, and large randomized clinical trials are being conducted to evaluate VD's chemopreventive efficacy against colon cancer in humans, but the mechanism underlying VD's anti-colon cancer activity remains to be defined. Secondary bile acids (BA) are potent colon cancer promoters. VD is known to affect colonic secondary BA levels by regulating host and colonic bacterial BA metabolism. Therefore we hypothesize that VD regulation of secondary BA levels is a key molecular mechanism underlying VD's chemopreventive activity against colon cancer. Moreover, the tumor-promoting secondary BA lithocholic acid (LCA) can also activate the vitamin D receptor (VDR), and LCA-VDR signals, like VD-VDR signals, also promote secondary BA catabolism by inducing Cyp3A, which can paradoxically limit LCA's tumor-promoting effects. Therefore we also hypothesize that VDR signals activated by VD or LCA limit secondary BA levels, thus mitigating tumor promotion by BAs. We will test these hypotheses using colon cancer models in genetically engineered mice with altered vitamin D hormone or VDR levels, and in gnotobiotic mice with altered bacterial secondary BA production. In Aim 1 we will determine the VDR-dependent host and microbe mechanisms that regulate Western diet-induced secondary BAs and colonic tumorigenesis. We will assess secondary BA levels and colonic tumorigenesis in WT, VDR(-/-) and transgenic mice over-expressing epithelial VDR that are fed Western diet or VD supplemented diet. In Aim 2 we will dissect the role of VD- independent LCA-VDR signals in colonic tumorigenesis. We will compare effects of Western diet or dietary LCA on secondary BAs and colonic tumorigenesis in WT, VDR(-/-), Cyp27b1(-/-) and VDR(-/-)/Cyp27b1(-/-) mice. Cyp27b1(-/-) and VDR(-/-)/Cyp27b1(-/-) mice cannot synthesize VD hormone, allowing us to dissect the chemopreventive effects of LCA-VDR signals without confounding effects of endogenous VD hormone. In Aim 3 we will assess the role of microbial 7-dehydroxylase (7-DH) in VD chemoprevention. We will compare tumorigenesis in gnotobiotic mice mono-associated with Lactobacillus acidophilus lacking 7-DH to mice also associated with Clostridium scindens expressing 7-DH fed Western diet or diet supplemented with VD. These studies will greatly advance our understanding of chemopreventive mechanisms of VD against colon cancer.